Myelodysplastic syndromes (MDS) are characterized by incompetent hematopoiesis that leads to single or multi-lineage peripheral cytopenias with the development of acute myeloid leukemia (AML) in approximately 30-40% of cases. With similarity to other cancer patients, MDS patients have defects in proximal T-cell receptor signaling pathways and altered T-cell homeostasis induced possibly by advanced age and a cancer-associated immunosuppressive microenvironment. A new class of therapeutic drugs (IMiDs), derived from the parent compound thalidomide possess a unique ability to augment T-cell function by substituting for inadequate secondary antigen-independent co-stimulatory signals through an unknown mechanism that involves activation of the CD28 receptor. These results gave rise the hypothesis that reversal of the T-cell signaling defects and improved T-cell homeostasis with lenalidomide along with a cellular vaccine will prevent leukemia progression in MDS. Since initiating the grant, we have conclusively shown that lenalidomide suppresses the phosphatase PP2A. Importantly, we demonstrate that lenalidomide is associated with an age- dependent increase in naove CD4+ T cells that is associated with the pro-T cell response after lenaldiomide treatment. Given the age dependency, these results suggest that lenalidomide stimulates CD4+ naove cell release from the thymus and thus blocks a target that is involved in thymocyte development. The goal is to decipher the complex molecular mechanism of lenalidomide during T cell development and confirm that the drug improves vaccine responses in vivo. However, there are obvious limitations since the necessary experiments must be performed in vivo. To overcome this problem, we now propose supplemental experiments using a "humanized" mouse model created by engraftment of human tissues including hematopoietic stem cells (HSCs) and/or T cells into immunodeficient mice (Prkdcscid IL2r3-/- (NOD/Shi-scid IL2r-/- or NOG). To extend our studies on lenalidomide and cancer vaccines, we propose three new supplemental aims using this mouse model, 1) to determine if thymic function is necessary for naove production and lenalidomide-induced function in CD4+ T cells, 2) to determine if PP2A alters the molecular checkpoint for naove T cell generation and 3) to determine if vaccine administration in combination with lenalidomide modulates antigen-specific T-cell response against foreign and tumor-associated antigens in a xenograft transplantation model of high-risk MDS. The supplemental aims are highly novel and innovative with tremendous implications for the future advancement of science. This application meets the definitions and goals of the NIH recovery act for competitive revisions since the revised application fits the overall scope of the parent project (i.e., development of a therapeutic vaccine in MDS), the project will provide rigorous evaluation for a novel treatment to be applied to a specific human disease (i.e., MDS), the work proposed can be accomplished within a two-year period, the work will generate a novel resource (i.e., xenograft model applicable for future drug discovery efforts in MDS), increases hours for part-time staff, enables the hiring of new staff, retains current personnel, and contracts new key personnel with important skills necessary to complete these new aims. PUBLIC HEALTH RELEVANCE: Myelodysplastic syndromes (MDS) are characterized by defective blood formation and high risk for leukemia development and primarily occur in individuals over the age of 65 years old. New strategies of treatment are needed for age-related diseases such as MDS as the US population ages. For tumor vaccine therapies to produce clinical responses in MDS and in cancer patients, appropriate antigen selection, intact antigen presentation, and T-cell function are all critical. We propose a new humanized mouse model in which to study specific questions generated from preliminary data in the parent grant application to decipher the combined effects of lenalidomide and cellular vaccines. We believe that this new treatment strategy is best tested in the setting of high-risk MDS that generally have poor survival, limited treatment options, and who may have a clinical response to the drug alone. Mechanistic studies will aide our understanding of T-cell immunity and improve our ability to utilize this form of immunotherapy and other forms for the treatment of cancer in general. The supplemental work fits the overall scope of the parent project (i.e., development of a therapeutic vaccine in MDS), the project will provide rigorous evaluation for a novel treatment to be applied to a specific human disease (i.e., MDS), the work proposed can be accomplished within a two-year period, the work will generate a novel resource (i.e., xenograft model applicable for future drug discovery efforts in MDS), increases hours for part-time staff, enables the hiring of new staff, retains current personnel, and contracts new key personnel with important skills necessary to complete these new aims.